The use of mass spectrometry in functional proteomics studies is a powerful approach for identifying proteins involved in important intracellular signal transduction systems. We propose to develop and use a new functional proteomics method based on isotope-coded affinity tag (ICAT) chemistry and liquid chromatography/mass spectrometry to identify proteins that participate in signal transduction complex formation in response to ligand stimulation. This strategy should improve the accuracy of relative protein quantitation as well as increase the speed, sensitivity, and dynamic range of protein identification over strategies based on the identification of proteins from SDS-PAGE gels. Ephrins and ephrin receptors (EphRs) are involved in patterning of the central and peripheral nervous systems. We will use our method to study signaling through EphRs as well as "reverse signaling" through B ephrin ligands in cultured neuronal cells by identifying and quantitating proteins that associate with previously characterized signal transduction proteins in response to stimulation by ephrinB2 or EphRs. Three groups of proteins will be identified: constitutively binding proteins, those that increase and those that decrease in their association with other signaling proteins in response to ligand stimulation. The goal of the proposed ICAT-based functional proteomics technology is to rapidly discover new protein-protein interactions that depend on ephrin signaling. A comprehensive cataloging of these interactions, especially including quantitative information and dependence upon the signaling state of the cell, will enable focused, hypothesis-driven experiments to elucidate the roles of these proteins in ephrin signaling. The functional proteomics methodology developed for the study of ephrin signaling could also be used to study a variety of other signal transduction systems essential for the functioning of the nervous system. Ultimately, information gained from these experiments may lead to greater understanding of the causes of a variety of cancers and neurodevelopmental diseases.